Bilirubin is the metabolic product of hemoglobin which is an oxygen carrier in blood. Determination of the amount of bilirubin in body fluid, especially blood, is important for detection of hemolysis and for checking liver function. One symptom of excess bilirubin in blood is jaundice. Determination of the bilirubin content in blood has been an important item of clinical chemical testing, and diazotization is the most common method of determination. In the diazotization method, bilirubin is coupled with diazonium salt such as diazosulfanilic acid and the amount of the resulting colorant is measured in a spectrophotometer to estimate the bilirubin content. Details of the diazotization method are described in J. B. Landis and H. L. Pardue, Clinical Chemistry, 24 (10), 1690-1699 (1978).
Bilirubin is a yellow colorant which itself has an absorption peak at 435 nm or 465 nm and a molecular absorptivity coefficient of about 5.times.10.sup.4 C.sub.33 H.sub.36 O.sub.6 N.sub.4. In hospitals and other medical establishments it is common to measure the bilirubin content by direct colorimetry wherein a sample of blood serum is put in a capillary or cuvette and its color density is directly read. Using the absorption characteristics of bilirubin, this direct colorimetric technique is described in several publications such as U.S. Pat. No. 3,569,721. The technique is used advantageously in the diagnosis of jaundice in infants because there is no need to subtract a predetermined corrective value from the measured value, which in the case of samples of adult serum is interfered with by yellow colorants other than bilirubin.
In accordance with a known method, the bilirubin content is detected by examining the density of green color formed with bilirubin is oxidized by an oxidizer to biliverdin and by knowing precisely how this density is proportional to bilirubin content. This method is called the oxidation method, and is described in prior art references such as U.S. Pat. Nos. 3,348,920 and 3,607,093. The method is suitable for use in qualitative testing, but a modification adapted to semiquantitative determination has also been proposed. Whether it is adapted to qualitative or semiquantitative determination, the oxidation method is used in qualitative testing of urine necessary for diagnosis of high-conjugation bilirubinemia. For details of these methods for quantitative determination of bilirubin, see Rinsho Kagaku Bunseki (Analysis in Clinical Chemistry), ed. by Masayuki Saito, II, pp. 248-279. Since all of these methods use a reaction in solution, they are generally referred to as the "solution method".
Recently, various dry methods for determination of the bilirubin concentration have been proposed. According to one such method, an absorbent base such as filter paper is impregnated with a stabilized diazonium salt, and after drying, a sample of serum is spotted on the test paper to determine the amount of color formation. The determination of bilirubin content by this technique is disclosed in, for example, Japanese Patent Application (OPI) Nos. 60591/74 and 99091/74 (the symbol "OPI" as used herein means an unexamined published Japanese patent application). However, since the bilirubin content in the blood serum of a healthy subject is very small, less than 1 mg/dl, it has been difficult to achieve accurate determination of the bilirubin concentration using a paper type testing device.
However, this method achieves a quick and simple determination of the bilirubin content. Therefore, it has been generally used as a semiquantitative method for primary screening in emergency or group examination, as well as for grouping of suspects.
Japanese Patent Application (OPI) No. 89796/78 (corresponding to U.S. Pat. No. 4,069,017) discloses a method for the quantitative determination of bilirubin with a multilayer film for chemical analysis. In this method, techniques are utilized for mordanting bilirubin with a mordant composition and shifting the absorption wavelength and increasing the molecular absorptivity coefficient by at least 50%. Other techniques are useful for eliminating interference by substances other than the bilirubin and these techniques are combined with techniques for forming a uniform layer of the reagent on a film. This makes possible the uniform spreading of a sample solution by a spreading layer, and by so doing, the method achieves high accuracy anaylsis and sensitivity that has not been attainable by the paper type testing device described above.
Attempts to perform quick and simple determination while achieving high accuracy and sensitivity are also described in Japanese Patent Application (OPI) Nos. 89797/78 (corresponding to U.S. Pat. No. 4,069,016) and 24694/80 (corresponding to U.S. Pat. No. 4,204,839). The method disclosed in Japanese Patent Application (OPI) No. 89797/78 takes advantage of the ability of bilirubin to be bound firmly to albumin or the like. According to the theory of this method, a complex of colorant and albumin is formed on a hydrophilic binder, the molecule of the colorant is replaced by a free bilirubin in a sample solution and the colorant released is measured by spectrometry.
The method described in Japanese Patent Application (OPI) No. 24694/80 detects the bilirubin mordanted by a material similar to that described in Japanese Patent Application (OPI) No. 89796/78. However, it measures the intensity of fluorescence generated by bilirubin upon irradiation with excitation light, rather than measuring the color density in the region of its absorption wavelength. In the methods of Japanese Patent Application (OPI) Nos. 89796/78 and 24694/80, the mordanting of the interacting polymer mordant for bilirubin is essential. One problem in the application of the polymer mordant is that its mordant effect is easily lost since the active site (quaternary N.sup..sym.) is apt to be neutralized with an acid, activator or neutral salt during application of a coating solution and formation of other layers on the mordant layer. To solve this problem, acetone, alcohol and other organic solvents must be used as a solvent for the coating solution of polymer mordant. Furthermore, the use of an anionic activator, acid, or dissociative salt must be avoided in forming a radiation-blocking layer or other functional layers on the mordant layer. Placing a bilirubin-containing sample solution on a multilayer film for chemical analysis having the polymer mordant also has difficulties. Since the sample solution contains not only bilirubin but also many other substances that can be mordanted, competition between bilirubin and these substances for adsorption onto the mordant often results in low adsorption capacity and a false photometry. Another disadvantage exists in that the known multilayer film for chemical analysis are not adapted for use with whole blood, so serum must be separated from a sample of whole blood before it is subjected to analysis.
Japanese Patent Publication No. 28119/78 (corresponding to DT-OS 2,240,357) discloses a method for preparing a test paper for detection of bilirubin in the body fluid. The method is based on fact that addition of a phosphate diester as well as a diazonium salt accelerates the rate of color-forming reaction appreciably due to the binding of the diazonium salt to bilirubin.
The most accurate method for quantitative determination of bilirubin that has been used for many years relies on the colorimetric analysis of an isolate of bilirubin extracted from an aqueous solution with a hydrophobic organic solvent such as chloroform, benzene, carbon disulfide and chlorobenzene. This method uses the property of these organic solvents to dissolve bilirubin adequately so that it can separate easily from an aqueous liquid sample in layers upon standing after it is mixed with the sample. However, in view of the current demand for a quick and simple method of blood analysis that requires a minimum of blood sample and still provides accurate measurements, the method which relies on direct extraction of bilirubin from an aqueous solution and requires a fairly large amount of sample is not highly recommendable.